ECD of PepT1 interacts with TM1 to facilitate substrate transport

Abstract

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of a wide variety of di- and tri-peptides and are essential for the absorption of dietary peptides in the digestive tract and the recovery of peptides in renal filtrate. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has 12 transmembrane (TM) helices that fold into two domains, the N-terminal domain (NTD, TM1-6) and C-terminal domain (CTD, TM7-12), and a large extracellular domain (ECD) of ~200 amino acids between TM9 and TM10. It is known that peptide transport involves large motions of the N- and C-domains, but the role of ECD remains unclear. Here we report the structure of PepT1 from Equus caballus (horse) determined by cryo-electron microscopy. The structure shows that ECD interacts with TM1 and bridges the N- and C-domains. Deletion of the ECD or mutations to the TM1-ECD interface both impair the transport activity. These results demonstrate a role of ECD in structure and function of PepT1 and enhance our understanding of the mechanism of transport in PepT1.

Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition

“…In the last decade, over 50 entries of the POT family were deposited in the Protein Data Bank (PDB), representing eleven bacterial and two mammalian homologs, bound to eight unique natural di-and tripeptides (Ala-Phe, Ala-Glu, Ala-Gln, Ala-Leu, Phe-Ala, Phe-Ala-Gln, Ala-Ala-Ala, alafosfalin), peptidomimetic drugs (valaciclovir, valganciclovir, 5-aminolevulinic acid) and a potent transport inhibitor Lys[Z(NO2)]-Val [3][4][5][6][7][8]10,18,[31][32][33][34][35][36][37][38][39][40][41] . These efforts revealed the basic principle underlying peptide binding in POTs which can be described as an electrostatic clamping mechanism between the invariable part of peptides (N-and C-termini as well as the peptide backbone) and conserved residues in the transporter (mainly via arginine, lysine, glutamate, asparagine and tyrosine residues).…”

MFSD1 in complex with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes

“…Members of the SLC15 family of proton-coupled oligopeptide transporters (POTs), namely SLC15A3 (PHT2) and SLC15A4 (PHT1), are localized to the lysosomal membrane. These two lysosomal SLC15 members share structural similarities with their plasma membrane-localized relatives, SLC15A1 (PepT1) and SLC15A2 (PepT2), and bacterial POT members that have been extensively characterized on a structural and functional level (Custodio et al, 2023;Killer et al, 2021;Kotov et al, 2023;Newstead, 2017;Parker et al, 2021;Shen et al, 2022). Overall, they all share the major facilitator superfamily (MFS) fold as well as typical POT signature motifs that are implicated in proton-coupling and peptide translocation (Aduri et al, 2015;Doki et al, 2013;Jensen et al, 2012;Parker et al, 2017;Solcan et al, 2012).…”